The most important player in the calcium induced calcium release process is the "cardiac" ryanodine receptor, RyR2. This is a large protein (500 kD) which forms a tetrameric channel 30x30x15 nm in size. The trans-membrane channel is believed to be formed by the C-terminal. Using standard approaches, RyR2 null mice produce an embryonic-lethal phenotype. We have therefore established a tripartite mouse model system to study the in-vivo function of this channel using a transgenic approach to produce an inducible, tissue-restricted null background by in adult mice hearts. To avoid embryonic lethality, we prepared several transgenic lines that would permit conditional and inducible gene targeting, limited to specific cardiac lineages (e.g. ventricular myocytes) and inducible at a desired developmental stage (particularly in the adult). The tools to accomplish this were the Cre recombinase - LoxP recombination system and the tetracycline trans-activator system. Three transgenic mouse lines were necessary to obtain spatial and temporal control of an inducible knock-out system. One mouse carries a Cre recombinase transgene under control of a tetracycline-sensitive promoter. The second mouse contains a lineage-specific promoter (alpha myosin heavy chain or myosin light chain 2v promoter driven tetracycline tranactivator), so that a tissue-specific knockout can be made to occur at a specified time. The third mouse contains a targeting construct with LoxP sites flanking an exon of the RyR2 endogenous gene. Using the reporter ROSA26 mouse, we have determined that cardiac-restricted expression of Cre Recombinase has indeed been achieved and that such a system is regulated by doxycycline/tetracycline. The mutant mice containing the RyR2 targeting vector are now being crossed with the regulatory transgenic mice to permit regulated knock-out of the transgene in the adult mouse. Their phenotype, both of individual cardiac myocytes and at a whole animal level, will be examined to determine the essential role of the RyR2 protein on EC coupling. Finally, rescue of the null-phenotype will be achieved through targeting of the floxed sequences with chimeric cDNA constructs to permit the endogenous transcriptional machinery of the RyR2 to transcribe mutant forms of the protein. Proof of this system has already been generated in ES cells null for the RyR2 gene, and during the coming year, we hope to exploit this system to generate transgenic mice with mutant forms of the ryanodine receptor to study specific questions related to the structure and function of the RyR2 protein.